The present invention relates to packing materials for liquid chrmatographic or catalytic columns, and more particularly it relates to an improved packing material for use in high performance liquid chromatography and to a method for making and using such a packing material.
Liquid column (LC) packing materials are usually porous materials which possess adsorptive or catalytic sites on the pore walls. They may be used as packings in columns or as loose material in vessels. LC packing materials typically are porous particles. However, they may instead be fibers or membranes. Porous membranes also provide filtration. When the pore size of the adsorptive or catalytic membrane excludes large proteins, then ultrafiltration is combined with adsorption or catalysis. Membranes may have small pores throughout their mass which exclude protein. Alternatively, the membrane may be a composite of sintered or adhered porous particles, in which case the pores between particles are large whereas the pores within the sintered or adhered particles are small. For example, Kontes Glass Co. markets a thick porous membrane of polyvinyl chloride upon whose large pore walls are attached small porous silica particles. The pores of the silica particles are much smaller than the pores upon whose walls the silica particles are attached.
Liquid column chromatographic techniques are used for the separation, analysis, and purification of small molecules as well as of polymers such as proteins in solution. Such separations are mediated either by surface interactions or by size or electrostatic exclusion interactions. Surface-mediated separations require a degree of adsorption of solute to the packing surface. The adsorption is usually due to physisorption and can be driven by hydrophobicity for the case of lipophilic solutes (reverse phase or hydrophobic interaction chromatography), by ion exchange for the case of charged solutes, and by bioaffinity interactions. Often several mechanisms occur simultaneously though one usually dominates. The adsorption can also be due to covalent bond formation to the support. For example, dissolved saccharides can form a covalent boronate bond to phenylboronic acid immobilized on a support.
Analysis and purification of biological fluids represents a particularly important application of liquid chromatography. Such biofluids include blood, plasma, serum, urine, tissue extracts and fermentation and cell cultures. Such biological fluids are generally highly aqueous with minimal organic cosolvent content. It is often advantageous to maintain a highly aqueous state since addition of organic cosolvents reduces the solubility of many such proteins, and hence can cause precipitation and loss of some components prior to chromatography. In other instances, precipitation is used to remove interfering substances.
Virtually all proteins are strongly adsorbed by reverse phase packings when the mobile phase in the column is weakly eluting, as is the case with highly aqueous mobile phases. Salting out effects can accentuate such adsorption. All proteins are also adsorbed by ion exchange packings when their charge is counter to that of the ion exchange sites on the packing surface. This condition can be attained by adjusting the mobile phase pH to the appropriate side of the isoelectric point (IEP). Proteins are also known to be adsorbed by unbonded chromatographic silica.
Size exclusion chromatography requires minimal adsorption to the packing surface. Such surfaces are typically very hydrophilic when proteins are subjected to size exclusion chromatography using highly aqueous mobile phases containing little or no organic cosolvent.
Liquid chromatography using a reverse phase packing has been found to be an effective tool in both qualitative and quantitative analysis for drug substances in blood, serum, plasma or urine. Typically the reverse phase packing material is made up of bonded alkyl silica and most typically the packing is a porous silica having octadecylsilane (ODS) bonded to it.
Although the efficiency of such packing materials is good, they have a limited life. While ODS packings absorb the lipophilic drug substances from the sample, they also absorb proteinaceous substances which tend to interfere with fractionation of the drug substance from other materials contained in the sample. This eventually leads to a complete fouling of the chromatographic column. Therefore, it has previously been necessary to carry out a preliminary sample preparation procedure to remove the troublesome proteins.
In the most conventional way, the proteins are precipitated, the aqueous supernatant is extracted with a water-immiscible organic solvent, the organic solvent is removed from the extract by evaporation, and the analyte residue is reconstituted in mobile phase before analysis by high-pressure liquid chromatography. This method is very time-consuming and cost-inefficient.
A second method currently employed involves the adsorption of analytes onto a reverse phase packing of octadecylsilane bonded to silica in a small disposable column. Although this technique can be automated, the columns can be used for only one sample because proteins remain on the packing, and as a result the technique is also cost-inefficient for multiple samples.
In a third method, a reverse phase packing of octadecylsilane bonded to silica is introduced into a precolumn, which is separated from, but connectable to, an analytical column by a switching valve arrangement. Serum samples are injected directly into the precolumn, where the proteins are denatured and accumulated, and the deproteinated analyte solution is passed into the analytical column for fractionation. After approximately three injections, the precolumn must be backflushed to remove the protein residue. This interruptive backflush is time-inefficient for a large number of samples. Furthermore, the octadecylsilane packing eventually deteriorates because proteins cannot be completely removed therefrom.
Accordingly, for reverse phase liquid chromatography it would be desirable to have a packing material which is less protein adsorptive. In my U.S. Pat. Nos. 4,773,994, 4,778,600, 4,782,040, 4,950,634 and 4,950,635 there are disclosed improved reverse phase packing materials, termed dual zone materials. The dual zone reverse phase packing materials display a reduced degree of serum protein adsorption due to a lipophobic fluoroalkyl phase in the external zone. The pore size of the packing material is small so that size exclusion prevents the protein from reaching the internal zone where the lipophilic partitioning phase retains and separates drug substances. Although the lipophobic phase reduces protein adsorption when the mobile phase contains greater than or equal to 20 percent organic cosolvent, further minimization of protein adsorption would be desirable, especially when using more highly aqueous mobile phases in the column.
Other approaches to achieving a packing which has an exterior non-adsorptive to proteins combined with a size-excluded reverse phase interior are known. Size-excluded enzymes have been used to selectively modify the exterior of silica bearing covalently bonded oligopeptides. See, e.g., I. H. Hagestam et al, "Internal Surface Reverse Phase Silica . . . ", J Chrom. 351, (1986) p. 239. However, the scope of choices for the internal partitioning phase is severally constrained since many desirable partitioning phases may not be easily embodied in an oligopeptide while still remaining cleavable by an enzyme.
An approach that eliminates most constraints on the internal partitioning phase is to coat the packing with sufficient protein to prevent further protein adsorption. When large amounts of serum albumin or plasma are loaded onto an ODS-silica column, the column adsorbs no further protein and is said to be saturated. The silica is selected to have a pore size that excludes the protein from the pores so that the internal reverse phase remains unfouled and separatively active towards small lipophilic solutes such as drugs in plasma. However, the coating is removed by strongly eluting mobile phases. Hence the column saturation is lost during periodic column cleanup or during gradient elution chromatography.
Most of the coating can be permanently attached by passing 100% methanol through the column to denature and physically crosslink the coating. However, some saturation is lost after applying this crosslinking method, so that the entire treatment must be performed several times. After several cycles of saturation followed by denaturation, a permanently saturated column results. Such columns have been used to directly inject plasma and serum samples for LC analysis of drugs. See, e.g., H. Yoshida et al, "Some Characteristics of a Protein-Coated ODS Column . . . ", Chromatographia, Vol 19, 1985, pp 466-472.
These columns also have significant disadvantages. The Height Equivalent Theoretical Plate height rises more than 75 micrometers after this treatment. Even for the relatively inefficient 26 micrometer ODS-silica used, the plate height rise caused an efficiency loss of over 70%. Smaller silica particles would display much greater efficiency loss if the same plate height rise occurred, as expected if the rise was due to the diffusional barrier of the coating. However, the cause of this relatively low efficiency has not been proven in the literature.
Simple calculus shows that the volume fraction due to the shell of coating relative to the entire coated packing is given by 6 t/D, where t is the coating thickness and D is the silica particle diameter in micrometers. Hence, the coating thickness is given by W*D/(600.alpha.), where W is the protein weight percent and .alpha. is the ratio of the coating volumetric density to that of the support.
Although not deemed in the literature to be very high, the amount of protein in the saturated solvent-stable column of Yoshida was found to be very high when compared to the support particle diameter. The product of weight percent times diameter was 2.5.times.26=65. Given that the bulk densities of protein and of porous silica are about equal, the data could suggest that a thick coating formed on the order of 1100 angstrom thick. Since a single albumin molecule is approximately an 80 angstrom diameter sphere, a multilayer coating may have resulted. Thick coatings are known to degrade efficiency by creating a large barrier to solute diffusion. See, e.g., Kirkland, J., "High Speed Liquid-Partition Chromatography With Chemically Bonded Organic Stationary Phase", Journal of Chromatographic Science, Vol. 9 (1971) pp. 206-214. Thus it appears that there is a need to obtain a permanently saturated but thin protein coating on supports.
A second approach to imparting a crosslinked protein coating onto packing materials employs simultaneous contact of glutaraldehyde with a concentrated solution of protein in an unbonded silica slurry in water. Such coated supports have high immobilized protein context and are useful for chromatography of dissolved protein. The object of this approach is to maximize the amount of immobilized protein short of creating an impermeable composite through which liquid could not readily flow. In this approach, the weak adsorption properties of the immobilized protein in the packing material are useful. See, e.g., M. Tsuboi et al, "Chromatography Carrier", Japanese Patent Application No. 198,334/85, Sep. 7, 1985. A similar method uses a two-stage glutaraldehyde crosslinking procedure in which the crosslinking was interrupted after a period of time by washing away serum albumin that had not yet deposited on the silica. Subsequently, more glutaraldehyde was added to ensure that the remaining albumin was tightly crosslinked and permanently attached to the silica. The two stage process ensured that large clumps of support particles were not glued together. Such clumps disrupt flow through the column and degrade efficiency. See, e.g., R. A. Thompson et al, ". . . Sorbents Obtained by Entrapment of Crosslinked Bovine Serum Albumin in Silica", Journal Chromatography, Vol. 465 (1989) pp. 263-270.
The two-stage crosslinking approach resulted in chiral packing materials useful for separating racemic mixtures. However, the efficiency for the isomers of benzoin was only 10,000 plates/meter (P/M). The expected efficiency for the 7 micrometer silica used is 30,000-40,000 P/M. The protein weight percent was 13% and 21% for silica whose pore diameters were 50 and 100 angstroms, respectively. The value of W*D of 119 could suggest that a very thick coating formed, which is consistent with the degraded column efficiency. Hence this approach is not favorable to attaining a saturated but efficient coated packing material.
Yet another approach to forming a protein coating is to use glutaraldehyde as a coupling agent in a first step by bonding it to an aminopropyl-silica, leaving an immobilized aldehyde residue to which in a second step protein can be bonded through the amino side chain of lysine amino acid residues. Often sodium cyanoborohydride or pyridine borane is used to stabilize the bond to the packing by reducing the intermediate imine to the secondary amine. It is common in a final step to block residual immobilized aldehyde by addition of an excess of some hydrophilic primary amine such as tris(hydroxymethyl)aminomethane, glycine, or ethanolamine to avoid non-specific bonding by aldehyde during affinity chromatography. See, e.g., F. R. Bernath et al, "Methods of Enzyme Immobilization", in Manual of Industrial Microbiology and Biotechnology, ed. A. L. Deman & N. A. Solomon, publ. Amer. Soc. Microbiology, Wash. D.C. (1986) pp. 244-5. This approach immobilizes protein by forming covalent bonds between it and the support. Although this approach yields usable products, the partitioning phase is limited in scope since it must also bear amino groups.
Column packing materials bearing biocatalytic residues are also subject to fouling by the sample or process fluid. Such fouling can be due to particulates or to large proteins and colloids in the process fluid. Fouling by particulates physically blocks the column or membrane. However, such fouling can be countered by backflushing. In contrast, fouling by proteins is difficult to reverse, particularly when the protein adsorbs to the packing exterior and obstructs the mouths of the pores. Consequently, obstruction of diffusion of solute to the catalytic interior reduces the activity. See, e.g., P. S. J. Cheetham, "Principles of Industrial Enzymology", Handbook of Enzyme Technology, ed. A. Wiseman, publ. J. Wiley (N.Y., 1985) pp 126-128. Reduction of protein adsorption to the packing material would alleviate problems due to this source of fouling.
Thus, despite all of the recent advances in HPLC packing materials, the need still exists for improved minimization of protein adsorption while possessing an internal adsorptive or catalytic phase so as to improve utility and extend the usefulness of such packing materials.